The bioremediation of groundwater aquifers and sediments contaminated with chlorinated aliphatic hydrocarbons (CAHs) depends on the activities of reductive dehalogenases that are present in some anaerobic microorganisms (Bouwer et al., (1983) Appl. Environ. Microbiol. 45: 1286-1294; DiStefano et al., (1991) Appl. Environ. Microbiol. 57: 2287-2292). Of particular importance are organohalogen-respiring bacteria, such as Dehalococcoides or Dehalogenimonas sp., because reductive dehalogenation is the only known mode of metabolic energy conservation in these microorganisms, and each group can carry up to 36 different non-redundant rdh genes (Seshadri et al., (2005) Science 307: 105-108; McMurdie et al., (2009) PLoS Genet. 5, e1000714; Moe et al., (2009) Int. J. Syst. Evol. Microbiol. 59: 2692-2697).
While organohalogen-respiring bacteria have been key for decontaminating polluted sites via biostimulation and bioaugmentation (bioremediation), there are many instances where such treatments have been hindered by the absence of key microorganisms and genes, enzymatic inhibition, hydrological complications, or incomplete management of microbial competition and associated biogeochemistry. Remediation of common groundwater contaminants such as tetrachloroethene (PCE), trichloroethene (TCE), 1,1,2-trichloroethane (1,1,2-TCA), and 1,2-dichloroethane (1,2-DCA) poses additional challenges since an appropriate assemblage of organohalogen-respiring bacteria, plus their supporting microbial communities, is required for complete dechlorination of these compounds to a harmless end product. Furthermore, it is unclear whether faithful representatives of the well-studied laboratory isolates are dominant organohalogen-respiring bacteria in sediments and groundwater, and to what extent their laboratory-studied phenotypes are relevant in the field.
Given this uncertainty, managing bioremediation of CAHs requires (i) gauging the structure of the microbial community, in particular the organohalogen-respiring bacteria; and (ii) being able to identify and differentiate between closely related but functionally distinct subpopulations. Such information is crucial for predicting and controlling the ecological responses of the microbial communities to natural or engineered perturbations during bioremediation. To be useful for both lab and field applications, any such molecular diagnostic for comprehensively quantifying organohalogen-respiring microorganisms and their complex rdh gene inventories should be simple, cost-effective, and require the minimum possible biological input material (Ziv-El et al., (2012) Biotechnol. Bioeng. 109: 2200-2210; Maphosa et al., (2010) Trends Biotechnol. 28: 308-316).
Metagenomics (Hug et al., (2012) BMC Genomics 13, 327), transcriptomics (Lee et al., (2012) Appl. Environ. Microbiol. 78: 1424-1436), proteomics (Rowe et al., (2012) Environ. Sci. & Technol. 46: 9388-9397), pan-genome-microarrays (Hug et al., (2011) Appl. Environ. Microbiol. 77, 5361-5369; Men et al., (2013) Appl. Microbiol. Biotechnol. 97: 6439-6450) and functional-gene tiling microarrays (Marshall et al., (2012) ISME J. 6: 814-826; Marshall et al., (2014) FEMS Microbiol Ecol. 86: 428-440) have been used to study the eco-physiology of organohalogen-respiring bacteria. However, these approaches have not been widely applied as tools in full-scale field studies due to the requirement of large amounts of DNA as input, bioinformatic complexity, cost constraints, and inadequate sensitivity of the assay primer pairs for detecting low-abundance genes in complex genomic backgrounds. A number of single quantitative PCR (qPCR) assay primer pairs targeting a few of the best understood rdh genes have been shown capable of overcoming these obstacles and are employed regularly in the remediation industry.